This study investigated the impact of chemical cleaning on the physicochemical properties of a nanofiltration membrane and its subsequent separation efficiency of inorganic salts and two pharmaceutically active compounds (PhACs), sulfamethoxazole and carbamazepine. Chemical cleaning was simulated by immersing virgin membrane samples in aqueous citric acid, sodium hydroxide (NaOH), ethylenediaminetetraacetic-acid (EDTA) and sodium dodecyl sulphate (SDS) at various temperatures for 18 h. The cleaning temperature did not exert any discernible impact on the surface charge of the NF270 membrane selected in this study. However, high cleaning temperatures were shown to either amplify or reduce the impact of chemical cleaning on several other membrane properties (including hydrophobicity, surface roughness and permeability) as well as the rejection of both inorganic salts and PhACs. The influence of chemical cleaning on the membrane surface roughness was enhanced at elevated cleaning temperatures. Similarly, at a high cleaning temperature, caustic and acidic cleaning caused a more significant increase in the membrane surface hydrophobicity than that at an ambient temperature. An increase in the cleaning temperature could also slightly amplify the decrease in the membrane permeability due to acidic cleaning. When a caustic cleaning solution (pH 11.5) was used, the membrane permeability only varied slightly with the temperature. Results obtained from Fourier transform infrared spectroscopy (FTIR) analysis suggest that chemical cleaning even at a high temperature did not permanently alter the chemical composition of the membrane active or support layer. Indeed, the effects of chemical cleaning at a high temperature on the physicochemical properties of the membrane could be attributed to the conformational changes of the membrane polymeric matrix. Chemical cleaning using citric acid, SDS or EDTA at a high temperature resulted in a considerable increase in the rejection of salts and PhACs in their neutral form. On the other hand, caustic cleaning at an elevated temperature had no discernible impact on the rejection of inorganic salts and neutral PhACs. This is because caustic cleaning and an elevated cleaning temperature cause opposing effects on the rejection of these solutes. Chemical cleaning at all temperatures investigated in this study did not affect the removal of negatively charged sulfamethoxazole.